Journal of Life Science Research
1
Online ISSN 2186-5809

J Life Sci Res 2017; 15:1-7Ⓒ 2017 Osaka Prefecture University

Rapid Communication

Association between the Porphyromonas gingivalis fimA type II genotype
and the nutritional intake of elderly women
Ayaka Yazawa,† Miki Maetani, Ayumi Takemura, and Shigeki Kamitani
College of Health and Human Sciences, Osaka Prefecture University, 3-7-30 Habikino, Habikino-City, Osaka 583-8555, Japan.
Received 3 October 2017; accepted 24 November 2017

The red complex bacteria Porphyromonas gingivalis (P. gingivalis), Tannerella forsythia (T. forsythia), andTreponema denticola (T. denticola) are major causative agents of periodontitis and are highly associated with
its severity. P. gingivalis exhibits a strong potential for adhesion and easily forms a biofilm. Its fimA genecan be classified by nucleotide sequence into six types. The P. gingivalis strains that possess type II fimA
genotype are predominant in periodontitis patients. Here we investigated associations among the P. gingivalis
fimA genotypes, periodontal disease infection in elderly women, and nutritional intake. We obtained samples
from the participantsʼ teeth and performed PCR using the primer specific for P. gingivalis fimA gene as atemplate and identified the bacteria through amplification of the 16S rRNA gene region. We amplified thefimA gene using primers specific to the six fimA genotypes. Dietary information over the previous 1‒2 months
was obtained via a questionnaire. P. gingivalis was detected in 97 % of the participants, in 49 % of which, theP. gingivalis fimA genotype was type II. Further, participants with the type II genotype had higher intakes ofvitamin D, niacin, vitamin B12, and fishery products and higher animal protein ratio in their diet than thosewith the other genotypes. The participantsʼ protein/energy ratio was within the recommended levels, but theiranimal protein ratio was higher than that of people of the same age, and particularly high in those with thetype II fimA genotype. The differences in intake suggest the presence of an association between nutritional
intake and P. gingivalis fimA genotype, although determining the exact reason for this shall require further
investigation.
Key words: Porphyromonas gingivalis; elderly women; fimA genotype; nutritional intake

1 Introduction
Periodontal disease is a chronic inflammation of thegums caused by pathogenic bacteria. In Japan, the prevalence of periodontal disease increases with age, affecting
53 % of the population aged 65‒70 years and 62 % of
those aged ≥ 70 years.1 Further, the proportion of people
aged ≥ 70 years with > 20 teeth is 37.1 %, which is lower
than that for the other age groups.2 Periodontal disease not
only causes the loss of teeth but is also associated withsystemic diseases, such as circulatory disease and diabetes.3
Therefore, the prevention and treatment of periodontal
disease are crucial. Bacteria known as the red complex
species, namely Porphyromonas gingivalis (P. gingivalis),Tannerella forsythia (T. forsythia), and Treponema denticola(T. denticola), are the major causative agents of periodontitis
and are highly associated with its severity.4 P. gingivalis†

Corresponding author, Email: ayazawa@rehab.osakafu-u.ac.jp

exhibits a strong potential for adhesion and easily forms a
biofilm. The P. gingivalis fimA gene can be classified into
six types (I, Ib, II, III, IV, and V) based on its nucleotide
sequences.5,6 P. gingivalis strains that possess type II fimA
are the most predominant in patients with periodontitis.7-9
There have been many studies on the risk factors of
periodontal disease, which report smoking and obesity as
potential risk factors.10-14 It has also been reported thatcalcium, foods with lactic acid bacteria, dietary fiber, andn-3 fatty acids are associated with periodontal disease.15-19
Moreover, female sex hormones are among the potential
factors modifying the rate of progression of periodontal
disease.20,21 However, very few studies have investigatedperiodontal disease infection in elderly women who
attended a lecture on health and its relationship with the
genotypes of P. gingivalis fimA, but none have examined
the association between these genotypes and nutritional
intake. In this study, we investigated associations between

2

periodontal disease infection in elderly women community
residents, the genotypes of P. gingivalis fimA, and thewomenʼs nutritional intake.
2 Material and Methods
2.1 Participants
The participants were 35 female (aged 69.1 4.7
years) general residents who participated in lecture events
for the prevention of life-style related diseases held at
Osaka Prefecture University. Lecture events were held
twice a year. In this lecture event, lectures were given on
diet and exercise for prevention of life-style related diseases. Elderly women who need nursing care and support
were excluded. In addition, those who are being treated ata dental office were excluded. Their masticatory performance was at the level appropriate for a normal diet. Theirmasticatory performance was evaluated using chewing gum(Masticatory Performance Evaluating Gum XYLITOL,LOTTE). We conducted this study with the approval of
the Ethical Review Board of the Osaka Prefecture Universityʼs Graduate School of Comprehensive Rehabilitation.
2.2 Plaque sampling and genomic DNA extraction
Dental plaque was collected from the participantsʼ
erupted teeth and subgingival with a sterile toothbrush for
1 min. The plaque adhering to the brush was removed bywashing several times in a test tube of sterile distilled water.
The plaque was collected by centrifugation at 1,600 g
for 20 min; the supernatant was discarded and the resultant
pellets were stored at ‒20 C until the DNA extraction.22
The genomic DNA of each sample was extracted using the
Wizard Genome DNA Purification Kit (Promega), and thesamples were stored at ‒20 C until use.
2.3 Detection of periodontal pathogens
The polymerase chain reaction (PCR) primers for
detecting the bacterial species used in this study are listed
in Table 1. PCR was initially performed with broad-range
eubacterial primers based on the bacterial 16S ribosom-

Table 1

al-RNA gene.23 All primers were purchased from Invitrogen, Japan. The PCR reaction mixture included 0.25 U
KOD FX Neo polymerase (TOYOBO), 0.2 mM deoxynucleotide triphosphates (dNTPs), polymerase buffer, 1 mM
primers, and 30 ng of DNA solution from the plaque as
the template DNA in a total volume of 25 µL. The samples were preheated at 95 C for 2 minutes followed by 25
cycles of amplification under the following conditions:denaturation at 98 C for 10 s, annealing at 55 C for 30 s,
and elongation at 68 C for 1 min using a T100 thermal
cycler (Bio-Rad). The 25 cycles were followed by elongation at 68 C for 5 minutes. The PCR products were confirmed by 1 % agarose gel electrophoresis and then purified
using the Gel/PCRTM DNA Isolation System (Viogene) ina unique final solution. A second nested PCR was
performed with specific primers for periodontal pathogens
designed on the basis of the 16S ribosomal-RNA gene
(Table 1), reported previously.24 The PCR reaction mixture
included 0.25 U KOD FX Neo polymerase, 0.2 mM dNTPs,
polymerase buffer, 1 mM primers, and 2 µL of purified
first PCR products as a template DNA in a total volume of
25 µL. The PCR conditions were the same as previously
described for the broad-range eubacterial primers except
for the elongation time, which was 30 s. Subsequently, 5µL of the PCR products obtained were mixed with 1 µL of
Ez-Vision One (AMRESCO) and electrophoresed through2 % agarose gel. The pathogen-specific bands were visualized under a UV light transilluminator.
2.4 PCR for the genotype analysis of P. gingivalis fimA
Table 2 shows the PCR primers for the genotype
analysis of the P. gingivalis fimA gene as described in previous reports.25 All primers were purchased from Invitrogen,Japan. The PCR reaction mixture included 0.25 U KOD FX
Neo polymerase, 0.2 mM dNTPs, polymerase buffer, 1 mM
primers, and 30 ng of DNA solution from the plaque as the
template DNA in a total volume of 25 µL. Samples weredenatured at 95 C for 5 min, followed by 35 cycles ofPrimer sets for detection of specific periodontal pathogen.

Primer
Eu-16S-S
Eu-16S-AS

Sequence
5’- GAG TTT GAT CCT GGC TCA G -3’
5’- AGA AAG GAG GTG ATC CAG CC -3’

Amplified size (bp)Pg-16S- S
Pg-16S- AS

5’- AGG CAG CTT GCC ATA CTG CG -3’
5’- ACT GTT AGC AAC TAC CGA TGT -3’

404

Tf-16S-S
Tf-16S-AS

5’- GCG TAT GTA ACC TGC CCG CA -3’
5’- TGC TTC AGT GTC AGT TAT ACC T -3’

641

Td-16S-S
Td-16S-AS

5’- TAA TAC CGA ATG TGC TCA TTT ACA T -3’
5’- TCA AAG AAG CAT TCC CTC TTC TTC TTA -3’

316~1,5003

denaturation at 94 C for 30 s, annealing at 58 C for 30 s,
and extension at 72 C for 30 s, with a final cycle of 72 C
for 7 min using the T100 thermal cycler. 5 µL of the PCR
products obtained were mixed with 1 µL of Ez-Vision
One and electrophoresed through 2 % agarose gel, withthe pathogen-specific bands visualized under a UV lighttransilluminator.
2.5 Physical measurements
We used a body composition meter (BC118-D,
TANITA) to measure the participantsʼ body composition.
Body mass index (BMI) was calculated by the standard
formula: BMI = weight (kg)/height (m2). BMI was divided
into three categories: underweight (< 18.5 kg/m2), normal(18.5‒24.9 kg/m2), and obese (≥ 25 kg/m2).26
2.6 Nutritional intake
Nutritional intake was determined through the Food
Frequency Questionnaire (FFQg version 3.0, Kenpaku Co.,
Ltd.), based on 29 food groups and 10 types of cooking.
This was used to estimate the energy and nutrient intakesof each participant during the previous 1‒2 months.

Table 2Detection ratio(%)

100

3 Results
Fig. 1 shows the results of the detection of periodontal
pathogenic bacteria. P. gingivalis was detected in 34 (97 %)of the participants, T. denticola in 28 (80 %), and T.
forsythia in all 35 participants. All three strains weredetected in 27 (77 %) of the participants. Fig. 2 shows the
proportion of each genotype of P. gingivalis fimA detected.
The sample collected from one participant was of a very
poor quality and was therefore excluded. The type II fimA
genotype was detected in 16 of the remaining 33 participants
with P. gingivalis; this was a significantly higher proportion
than for any of the other fimA genotypes (p < 0.001)

Primer sets for analysis of fimA genotype.

PrimerfimA-1-S
fimA-1-AS

Sequence
5’- CTG TGT GTT TAT GGC AAA CTT C -3’
5’- AAC CCC GCT CCC TGT ATT CCG A -3’

fimA-1b-S
fimA-1b-AS

5’- CAG CAG AGC CAA AAA CAA TCG -3’
5’- TGT CAG ATA ATT AGC GTC TGC -3’

271

fimA-2-S
fimA-2-AS

5’- GCA TGA TGG TAC TCC TTT GA -3’
5’- CTG ACC AAC GAG AAC CCA CT -3’

292

fimA-3-S
fimA-3-AS

5’- ATT ACA CCT ACA CAG GTG AGG C -3’
5’- AAC CCC GCT CCC TGT ATT CCG A -3’

247

fimA-4-S
fimA-4-AS

5’- CTA TTC AGG TGC TAT TAC CCA A -3’
5’- AAC CCC GCT CCC TGT ATT CCG A -3’

251

fimA-5-S
fimA-5-AS

5’- AAC AAC AGT CTC CTT GAC AGT G -3’
5’- TAT TGG GGG TCG AAC GTT ACT GTC -3’

462

97

Amplified size (bp)
392

Ⅴ
3%

100
80

80

Ⅳ
24%

60
40

Ⅲ
3%

20
0

Fig. 1

2.7 Statistical analysis
The chi-square test was used to evaluate differences inthe detection ratios of the periodontal pathogens. Independent t-tests were used for the comparisons of nutritional
intake and body composition according to the genotype of
P. gingivalis fimA, and a p-value of < 0.05 was considered
statistically significant.

P. gingivalis

T. forsythia

T. denticola

Periodontopathic bacterial species detected in theparticipantsʼ plaque.

Fig. 2

Ⅰ
21%

Ⅱ
48%

Type Ib: not detected.

Distribution of the five P. gingivalis fimA genotypes(n = 33).

4

Table 3 shows the physical characteristic data for theparticipants with P. gingivalis, comparing the 16 participantsin which the type II fimA genotype was detected, with theremainder. There were no significant differences between
the two groups. Overall, the mean BMI was 22.8 kg/m2,
with six participants having a BMI greater than 25 kg/m2.
The participantsʼ mean fat/energy ratio was within
the range of 20 ‒30 % energy indicated by the Dietary
Reference Intakes (DRIs) for Japanese,27 but the individual

Table 3values for 19 (54 %) of the 35 participants exceeded 30 %.
The protein/energy ratio was also within the range of 13‒
20 % energy indicated by the DRIs. Overall, the participants
did not meet the DRI for dietary fiber, a factor for which
an association with periodontal disease has been noted.
The mean calcium intake reached the DRI value, but the
intake of 10 (29 %) of the 35 participants was below the
DRI value.
Table 4 presents the results of the nutritional intake

Physical characteristics of the participants with P. gingivalis.

P. gingivalis fimA typeII
I, III, IV, and V(n = 16)(n = 17)Age (years)
70.1 ± 4.5
68.1 ± 4.9
Height (cm)151.3 ± 4.0
154.3 ± 3.8
Weight (kg)54.2 ± 9.4
53.1 ± 7.5
23.7 ± 4.1
22.3 ± 2.8
BMI (kg/m2)
Fat (%)
35.2 ± 6.9
30.7 ± 5.9
Values are mean ± SD. Type Ib was not detected.

Table 4

 he nutritional intake of the participants with P. gingivalis, comparing those withT
the type II fimA genotype with the remainder.

P. gingivalis fimA typeII
I, III, IV, and V(n = 16)(n = 17)
Energy (kcal)
2045 ± 418
1912 ± 445
Protein/energy ratio (%energy)
16.0 ± 2.5
14.6 ± 1.4
Fat/energy ratio (%energy)
30.2 ± 4.0
29.4 ± 3.7
Carbohydrate/energy ratio (%energy)
53.7 ± 6.0
56.0 ± 4.8
Protein (g)82.6 ± 23.4
70.3 ± 20.5
51.9 ± 6.1
Animal protein ratio (%)
57.6 ± 8.4＊
Calcium (mg)709 ± 185
681 ± 212
Phosphorus (mg)1232 ± 331
1101 ± 310
Iron (mg)
9.0 ± 2.3
8.0 ± 2.2
Magnesium (mg)294 ± 71268 ± 75
Zinc (mg)
9.4 ± 2.4
8.3 ± 2.1
Copper (mg)1.22 ± 0.24
1.13 ± 0.30
Retinol activity equivalents (μg)
710 ± 156659 ± 169
8.2 ± 3.5
Vitamin D (μg)
11.9 ± 4.9＊
α-Tocopherols (mg)
7.6 ± 1.7
6.9 ± 2.0
Vitamin K (μg)
264 ± 61252 ± 67
Thiamin (mg)1.12 ± 0.29
0.95 ± 0.26
Riboflavin (mg)1.28 ± 0.35
1.16 ± 0.33
14.9 ± 4.8
Niacin (mg)19.4 ± 7.0＊
Vitamin B6 (mg)1.40 ± 0.37
1.18 ± 0.32＊
Vitamin B12 (μg)
10.5 ± 4.4
7.3 ± 2.8
Vitamin C (mg)
125 ± 31114 ± 33
Folate (μg)
347 ± 67329 ± 82
Pantothenic acid (mg)6.31 ± 1.50
5.77 ± 1.57
Dietary fiber (g)16.0 ± 2.6
15.5 ± 4.2
Salt equivalent (g)10.6 ± 2.3
10.5 ± 4.0
Cholesterol (mg)366 ± 140309 ± 115
Potassium (mg)2864 ± 646
2602 ± 720
n-6 Fatty acids (g)10.79 ± 3.16
10.43 ± 3.47
n-3 Fatty acids (g)2.84 ± 0.94
2.30 ± 0.82
Values are mean ± SD. * p < 0.05. Type Ib was not detected.

5analysis for the 33 participants with P. gingivalis, showing
a comparison between the 16 participants with the type II
fimA genotype and the remainder. Those with the type II
genotype consumed a significantly higher ratio of animal
protein to total protein (p = 0.036) and had significantly
higher intakes of vitamin D (p = 0.012), niacin (p = 0.046),
and vitamin B12 (p = 0.019). The protein/energy ratio wasalso higher in those with the type II genotype, but this did
not achieve statistical significance (p = 0.056). There wasno significant difference between the groups for any othernutrient. In the analysis of food groups, those with thetype II genotype showed a greater intake of fishery products (p = 0.012).
4 Discussion
Elderly women examined in this study were participants who took part in lecture events for life-style relateddiseases prevention on their own will. Considering thisfact, participantsʼ awareness of health was not necessarily
low. Nevertheless, 16S rRNA gene of P. gingivalis was
detected in 97 % of the participants (Fig. 1), and the type II
genotype of fimA gene in P. gingivalis was found in 48 %of those who P. gingivalis detected women (Fig. 2). It hasoften been reported that the type II genotype is predominant
in patients with severe periodontal disease;7 we speculated
that the periodontal disease was possibly progressing inpeople infected with P. gingivalis with this genotype. The
relationship between the genotype and the virulence havediscussed in many reports from the pathogenic and molecularmechanistic point of view.28-36 However, the relationshipbetween the genotype and nutrient intakes was unknown
till now. Thus, we examined this relationship for elderly
women by using the food frequency questionnaire (FFQ)
method.
Comparing of nutrient intakes between the participants
with fimA type II genotype and with other types indicatedthat those with the type II genotype was significantly higher
intakes of vitamin D, niacin, and vitamin B12 (Table 4). In
the DRIs for Japanese, the tolerable upper intake level(UL) values were set for vitamin D and niacin. UL was set
to prevent adverse health conditions that would be causedby an excessive intake of certain nutrients. UL value ofvitamin D was 100 µg, and niacin was 250 mg (quantity as
nicotinamide) and 60 mg (quantity as nicotinic acid), but
the participants with fimA type II genotype did not reach
UL values (vitamin D: 11.9 4.9 µg, niacin: 19.4 7.0 mg
in Table 4). In the DRIs for Japanese, the protein/energy
ratio has tentative dietary goal for preventing life-style
related diseases (DG) value. DG value of protein/energy
ratio was 13‒20 % energy. The mean protein/energy ratioin participants of both groups met DG value (16.0 2.5 %energy, 14.6 1.4% energy in Table 4). Moreover, theanimal protein ratio of the participants (54.6 8.0 %) was
higher than that of women with similar ages (60‒69 years
old: 50.7 %, 70 years and over: 49.5 %).2 Notably, theanimal protein ratio of the participants with the type II fimA
genotype (57.6 8.4 %) was particularly high. To date,
what makes the difference of these nutrients between two
groups of the fimA genotypes remains in this study. Further
investigation is required for understanding the relationshipbetween these nutrients and fimA genotypes.
A previous study reported that periodontal disease
was more severe in women whose amount of daily calcium
intake was less than 500 mg compared to 800 mg or more.15
The calcium intake of both groups showed more than 500
mg and no significant difference between two groups (709
185 mg, 681 212 mg in Table 4). In addition, it isreported that benefits of higher intake of high-fiber foods on
slowing periodontal disease progression are most evident in
men aged 65 and older.18 In this study, the intake of dietaryfiber was almost equivalent for two groups (16.6 2.6 g,
15.5 4.2 g in Table 4). But, it is difficult to directlycompare the value of the intake between previous studies
and our present study because the diet survey method was
different among these studies. The intake of dietary fiberof participants in this study were less than DG value (50‒
60 years old: ≥ 18 g, 70 years and over: ≥ 17 g), suggestingthat it is desirable for both groups to increase the intake ofthe high-fiber foods. Furthermore, higher dietary intakesof n-3 fatty acids were associated with lower prevalenceof periodontitis.19 The n-3 fatty acids intake of the type IIgroup (2.84 0.94 g) was higher than other genotype group(2.30 0.82 g) in the study (Table 4) though the difference
between two groups was not significant, and both groups
met the adequate intake (AI) value, which indicates theamount adequate to maintain a certain level of nutritional
status, in the DRIs for Japanese. The result of these threenutrients was apparently inconsistent with previous reports
described above because the type II genotype was mainly
found in the patients with severe periodontitis. These reports
were studied based on western diet for western people.
This study was the result of Japanese food for Japanese.
Therefore, there was also a possibility that out differences.
For that reason, it may be necessary to consider diet styleand racial differences.
There are some limitations to this study. First, a dietsurvey was based on a self-report. Second, there may havebeen selection bias because participant attended a programof their choice. To clarify the relationship between the
nutrient intakes and the fimA genotypes, further study isrequired: for instance, whether the amount of these nutrient
affect the growth of P. gingivalis, the biofilm formation and6colonization to the cells depending on the fimA genotypes
in vitro and in vivo. Promotion of healthy nutrition andadequate physical activity may be additional factors to help
prevent or delay the progression of periodontal disease.37
A more detailed investigation into dietary habits will elucidate the association between the nutrient intakes and the
genotypes of P. gingivalis fimA gene, by fulfilling a new
study including various aged people with and without
P. gingivalis infection.

10

Acknowledgements
We thank all the participants and the local staff for
their involvement in this study.

12

11

13
Funding
This research did not receive any specific grant fromfunding agencies in the public, commercial, or not-for-profit
sectors.
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